Hemoglobin is a tetrameric protein which delivers oxygen via an allosteric mechanism. There are four binding sites for oxygen on the hemoglobin molecule, as each protein chain contains one heme group. Each heme group contains a substituted porphyrin and a central iron atom. The iron atom in heme can be in the ferrous (+2) or ferric (+3) state, but only the ferrous form binds oxygen. The ferrous-oxygen bond is readily reversible. The binding of the first O2 molecule to hemoglobin enhances the binding of additional O2 to the same hemoglobin molecule. In other words, O2 binds cooperatively to hemoglobin. Thus, binding of the first oxygen to a heme requires much greater energy than the second oxygen molecule, binding of the third oxygen requires even less energy, and the fourth oxygen requires the lowest energy for binding. Hemoglobin A, the principal hemoglobin in adults consists of two α and two β subunits arranged with a two-fold symmetry. The α and β dimers rotate during oxygen release to open a large central water cavity. The allosteric transition that involves the movement of the alpha beta dimer takes place between the binding of the third and fourth oxygen.
Using well-known equipment such as the AMINCO™ HEM-O-SCAN, an oxygen dissociation curve can be plotted to determine the affinity and degree of cooperativity (allosteric action) of hemoglobin. In the plot, the Y-axis represents the percent of hemoglobin oxygenation and the X-axis represents the partial pressure of oxygen in millimeters of mercury (mm Hg). If a horizontal line is drawn from the 50% oxygen saturation point and a vertical line is drawn from the intersection point of the horizontal line with the curve to the partial pressure X-axis, a value commonly known as the P50 is determined. This is the partial pressure (mm Hg) at which the hemoglobin sample is 50% saturated with oxygen. Under physiological conditions (i.e. 37° C., pH 7.4, and a partial pressure of carbon dioxide of 40 mm Hg), the P50 value for normal adult hemoglobin is around 26.5 mm Hg. If a lower than normal P50 value is obtained for the hemoglobin being tested, the oxygen dissociation curve is considered to be “left-shifted” and the presence of high affinity hemoglobin is indicated. Conversely, if a higher than normal P50 value is obtained for the hemoglobin being tested, the oxygen dissociation curve is considered to be “right-shifted” and the presence of low affinity hemoglobin is indicated. Such low affinity hemoglobin will lose oxygen more easily at lower pressures of oxygen, and therefore may be useful to deliver oxygen to tissues more efficiently.
It has been suggested that influencing the allosteric equilibrium of hemoglobin may be a viable method to treat diseases that are influenced by oxygen delivery. For example, the conversion of hemoglobin to a high affinity state is generally regarded to be beneficial in treating problems associated with deoxyhemoglobin S (sickle cell anemia.). The conversion of hemoglobin to a low affinity state is believed to be of general utility in a variety of disease states in which tissues suffer from low oxygen tension, such as ischemia, radio-sensitization of tumors, carbon monoxide poisoning, fetal oxygen delivery and the restoration of the oxygen affinity of stored blood.
FIGS. 1A-1D depict the chemical structures of a variety of compounds which have a “right-shifting” allosteric effect on hemoglobin (referred to herein as “allosteric hemoglobin modifier compounds” or “allosteric effector compounds”). The family of compounds represented by the general structure illustrated in FIG. 1D (referred to as “RSR compounds”), are representative of a large family of compounds having a strong allosteric effect. For example, one compound in this family, 2-[4-((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy]-2-methyl propionic acid (efaproxiral, also referred to as RSR13), which has the following structure, when X+ is H+:
is an allosteric effector of hemoglobin, and has been shown to enhance tissue oxygenation in vivo. In general, efaproxiral is administered as a physiologically acceptable salt, such as the monosodium salt; that is, X+ is Na+. Efaproxiral induces allosteric modification of hemoglobin, such that its binding affinity for oxygen is decreased, resulting in increased oxygen distribution to tissues by erythrocytes. Efaproxiral has been reported to enhance fractionated radiation therapy in mice bearing the Lewis lung carcinoma. See Teicher (1996) Drug Dev. Res. 38:1-11. Enhancement of the effect of radiation was observed in EMT6 mouse mammary tumors by treatment with efaproxiral plus oxygen breathing, with the absence of enhanced radiation effects in normal tissues. Rockwell and Kelley (1998) Rad. Oncol. Invest. 6:199-208. Additionally, mouse fibrosarcoma tumor growth has been shown to be reduced by the combination of efaproxiral and radiation relative to radiation alone. See Teicher (1996) Drug Dev. Res. 38: 1-11; Khandelwal et al. (1996) Rad. Oncol. Invest. 4:51-59. This family of compounds, together with their utility and methods for using them are described in a number of patents including, U.S. Pat. No. 5,661,182, issued Aug. 26, 1997, U.S. Pat. No. 5,290,803, issued Mar. 1, 1994, U.S. Pat. No. 5,382,680, issued Jan. 17, 1995, U.S. Pat. No. 5,432,191, issued Jul. 11, 1995, U.S. Pat. No. 5,648,375, issued Jul. 15, 1997, U.S. Pat. No. 5,677,330, issued Oct. 14, 1997, U.S. Pat. No. 5,731,454, issued Mar. 24, 1998, U.S. Pat. No. 5,122,539, issued Jun. 16, 1992, U.S. Pat. No. 5,927,283, issued Jul. 27, 1999, U.S. Pat. No. 5,827,888, issued Oct. 27, 1998, U.S. Pat. No. 5,049,695, issued Sep. 17, 1991, U.S. Pat. No. 5,591,892, issued Jan. 7, 1997, U.S. Pat. No. 5,049,695, issued Sep. 17, 1991, U.S. Pat. No. 5,250,701, issued Oct. 5, 1993, U.S. Pat. No. 5,248,785, issued Sep. 28, 1993, U.S. Pat. No. 5,705,521, issued Jan. 6, 1998, and U.S. Pat. No. 5,525,630, issued Jun. 11, 1996. Each of these references is specifically incorporated herein by reference in its entirety.
As a result of the general utility and importance of these compounds a number of methods have been developed to synthesize them. Two of the principal methods developed to date are compared in FIG. 2 using the synthesis of the sodium salt of 2-[4-((((3,5-dimethylphenyl)amino)carbonyl)methyl)phenoxy]-2-methyl propionic acid (also referred to herein as efaproxiral sodium and efaproxiral-Na) (5), for purposes of illustration. With reference to FIG. 2, in the first method developed (referred to as Process A), efaproxiral-Na (5) was synthesized as the free acid (6), which was then treated with base to provide the sodium salt (5). (Randad et al. (1991) J. Med. Chem. 34:752-757). In the second method (referred to as Process B), this compound was synthesized as the ethyl ester (4), which was then saponified to provide the sodium salt (5). (Witt et al., DE 2,432,560, published Jan. 22, 1976). Process A is highly exothermic, not easily amenable to commercial scale manufacture and uses a halogenated hydrocarbon solvent. Process B eliminates the use of a halogenated hydrocarbon solvent and is more amenable to commercial scale manufacture and thus is the preferred method. The primary drawback of Process B, however, is the unexpected generation of the polymeric impurity poly(ethyl methacrylate) and precursors to this compound, which are referred to herein collectively as PEM, which is formed in Step 2 via the following mechanism.

In the manufacture of the efaproxiral sodium (5) via Process B poly(ethyl methacrylate) is typically formed in concentrations of from approximately 0.5% (5000 parts per million (ppm)) to 9% (90,000 ppm) by weight.
Despite the general utility and importance of these compounds in treating disease, problems remain in generating pharmaceutical grade compositions. Specifically, the compounds are administered to patients in a sterile intravenous (IV) solution preparation. In the process of testing these compounds, difficulty with the drug product manufacturing (IV solution formation) has been traced to the PEM byproduct generated during their synthesis as outlined above. Thus, there is a need for methods to reduce the level of polymeric impurity in preparations of allosteric hemoglobin modifier compounds in order for use in patients. There also remains a need for compositions of allosteric hemoglobin modifying compounds with lower amounts of impurities in general. The present invention provides an improved process for making highly pure allosteric effector compounds.
Another problem associated with the prior art methods is that there is currently no effective way to measure the low levels of impurities capable of causing failure in the IV solutions prepared from compositions, which are comprised of these compounds. As noted above, a particularly undesirable impurity is PEM. Prior art methods for measuring PEM are deficient in several respects, particularly in that they are unable to detect very low levels of PEM. Current methods for detecting and measuring PEM include 1H NMR, gel permeation chromatography (GPC) or size exclusion chromatography (SEC); MALDI-TOF mass spectrometry; ultraviolet (UV) analysis; and infrared (IR) analysis. The latter two techniques are accurate for mixtures containing ≧2-4% PEM w/w in efaproxiral ethyl ester (4) or efaproxiral sodium (5). The former techniques can be used for determining the concentration of ≧0.5% w/w PEM. For example, to analyze intermediates with ≧0.5% w/w PEM, 1H NMR can be used by comparing the integration of the PEM methylene proton signal to the ethoxy-methylene proton signal of the efaproxiral ethyl ester (4). By multiplying the appropriate molecular weights to the respective signals of the PEM and efaproxiral ethyl ester one can develop a formula for determining the percent weight/weight (% w/w) of PEM. Muguruma et al. describe a method for the quantitative analysis of poly(methylmethacrylate) (PMMA) in drug substances using pyrolysis-gas chromatography (PY/GC). Using this method, Muguruma were able to detect levels of PMMA ≧0.1 wt % with a precision of approximately 4.5% at a level of 0.1%. (Mugurma et al. (July 1999) LC-GC International, pp. 432-436).
For analysis of highly pure compositions of allosteric hemoglobin modifiers however, none of the prior art techniques can be used because the limit of detection is not low enough. The improved processes of the instant invention produce compositions of allosteric hemoglobin modifiers having very low levels of impurities (≦100 ppm (0.0100% w/w) of PEM in efaproxiral-Na). Consequently, there remains a need for a method for analyzing these compounds which has a low detection limit and good specificity to measure very low levels of PEM, as well as other polymeric impurities with adequate sensitivity. Since pyrolysis/gas chromatography/mass spectrometry (PY/GC/MS) has been used for identification of polymers in relatively intractable matrices, it was evaluated to determine whether it would be useful for trace level analysis of polymers in efaproxiral-Na. Extensive development led to the discovery of a method for quantitation of trace levels of PEM (limit of quantitation=10 ppm). The PY/GC/MS method described herein is a novel analytical technique that utilizes single ion monitoring and an isotopically labeled PEM internal standard to provide the sensitivity, precision, accuracy and reproducibility required for the detection and quantitation of a trace level impurity. This technique can be extended to the analysis of compositions of allosteric hemoglobin modifiers containing polymeric impurities other than PEM in the event that the method of synthesis illustrated in FIG. 3 is modified.
It is therefore an object of this invention to provide compositions of allosteric hemoglobin modifying compounds having lower amounts of polymeric impurities, particularly PEM, as well as, lower amounts of impurities in general.
It is also an objective of the present invention to provide improved methods for the synthesis of compositions of allosteric hemoglobin modifying compounds having lower amounts of polymeric impurities.
It is another object of the present invention to provide improved methods for purification of compositions of allosteric hemoglobin modifying compounds prepared by any known synthetic method, in particular by the method disclosed herein.
Finally, it is an objective of the present invention to provide a method for analyzing compositions of allosteric hemoglobin modifying compounds, which enables detection and quantification of low levels of impurities, particularly polymeric impurities.